Method and apparatus for performing phase fluorescence lifetime measurements in flow cytometry

ABSTRACT

PCT No. PCT/US91/07259 Sec. 371 Date Apr. 12, 1993 Sec. 102(e) Date Apr. 12, 1993 PCT Filed Oct. 10, 1991 PCT Pub. No. WO92/07245 PCT Pub. Date Apr. 30, 1992.A method and apparatus for identifying individual particles or cells which have been labeled with different fluorochromes, on the basis of the lifetime of their fluorescence, or based on different decay times for a fluorochrome in different cells.

This application is a continuation-in-part of application Ser. No.07/595,343, filed Oct. 10, 1990, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a flow cytometry method and apparatusfor distinguishing and/or characterizing cells or particles on the basisof measured fluorescence lifetimes. More particularly, the presentinvention concerns a flow cytometry method and apparatus fordistinguishing and characterizing a particle or cell which has beenlabeled or associated with one or more fluorophores having lifetimeswhich are modified due to one or more characteristics or properties of acell or particle with which the fluorophore is associated, wherein thelifetime measurments are independent of the intensity of the detectedfluorescence emission.

Research involving the study and analysis of cells, generally known ascytology, employs a variety of analytical techniques for identifying andenumerating the subpopulations of cells in a specimen under study. Forexample, cytological materials may be examined to detect the presence ofcancerous or malignant cells, or characteristics of the cells within aspecimen. For purposes of analysis, the cells may be labeled with avariety of fluorescent materials, conventionally known as fluorophoresor fluorescent probes, which have an identified affinity for cells orcell components which are of interest to an analysis. The fluorophoreswill emit a particular fluorescence radiation when stimulated by lightat a wavelength corresponding to the excitation wavelength of thefluorophore. The wavelength and/or intensity of the emitted light hasbeen used to analyze a subpopulation of cells, wherein differentfluorophores can be used to distinguish subpopulations offluorophore-labeled cells.

The study of collections of multiple cells using fluorescentspectroscopy has obvious limitations. For example, an accuratedetermination of the number of cells in a subpopulation having a givencharacteristic cannot be made and, more importantly, the subpopulationscannot be separated for further analysis. In order to permit themeasurement and analysis of a population of cells (or any biologicalparticle such as isolated nuclei, chromosome preparations orneurobiological organisms) on an individual basis, fluorescence flowcytometry has been employed.

Fluorescence flow cytometry, which involves the intensity and/orwavelength measurement of fluorescence emissions from individual cellslabeled with a fluorophore while the cells are flowing in a liquid orgaseous medium past an observation point, permits analysis of individualcells as well as sorting of the cells based upon that analysis. Adescription of examples of fluorescence flow cytometry appears in"Practical Flow Cytometry", 2d. ed., H. Shapiro (1988), Liss & Co, theentire contents of which are herein incorporated by reference.

A conventional flow cytometer has a sample handling and delivery systemwhich collects the cell population into a stream of individual cellswhich is directed one cell at a time past the observation point of theflow cytometer. When a liquid medium is used, the stream containing thesamples is sheathed by a surrounding fluid stream to insure that onlysingle cells pass the observation point.

A conventional flow cytometer also has a parameter detection systemwhich can include a focused light source, typically a laser, thatdirects a narrow beam of light at a predetermined wavelength to theobservation point where an individual fluorophore-labeled cell passingthrough the point may be illuminated, resulting in a fluorescenceemission from the fluorophore label. The parameter detection system alsoincludes collection optics and optical transducers, such asphotomultipliers and detectors that receive the fluorescence emission atthe observation point and convert it to electrical signals which arerepresentative of the intensity and/or wavelength of the emitted light.

Where the cells have been tagged with a fluorophore having an affinityfor a particular characteristic or composition of the cell, as well asbeing excited at the wavelength of light emitted by the laser, the lightemission intensity and/or wavelength of the fluorophore bound to eachcell at the observation point may be detected for purposes of analysis.

When the fluorophore bound to a cell is excited by light at thefluorophore's absorption wavelength, the fluorophore's electrons canabsorb energy such that the energy level of an electron is raised fromthe ground state to an excited state. The excited electron then emits aphoton as it returns to the ground state, with the photon having acharacteristic wavelength and intensity.

In FIG. 1 is shown an advanced streak camera flow cytometry apparatus.The pulsed output from the light source 1 passes through an opticssubsystem 2 where it is shaped and focused into the continuous flow ofcells or particles. The cells are in a suspension from sample source 4and are aligned within sample chamber 5 by the laminar flow of a fluidsheath from a solution source 6. When the laser light illuminates anindividual cell at the observation point in a flow chamber 3, afluorescence is emitted as pulsed fluorescence radiation 7. This pulsedradiation is captured by an optics subsystem 8 and focused onto thestreak camera detector 9. The output of the detector is a voltage thatis applied by line 16 to a signal processing system 10 where it is usedfor cell population analysis based on intensity and/or wavelength of theemitted fluorescence. The signal processing is fast enough to controlthe division of the observed cells into subpopulation collections by aconventional cell sorter 11.

Thus, measurements of the intensity and/or wavelength of emittedfluorescence over a period of time may be used as the basis fordiscrimination and sorting of the cells or particles in the populationunder study, and the intensity of emitted light may be measured byphotomultipliers.

Several conventional methods for distinguishing cells by detecting theemitted fluorescence light at the observation point of a flow cytometersystem are known. The advanced streak camera system of FIG. 1 measuresfor each cell the change in the intensity of transient emitted lightover a period of time as the cell passed through the laser beam. Basedupon such measurements, the attenuation time of the emitted light, therise time of the emitted light and the orientation relaxation time maybe detected and used as a basis for cell discrimination. Referring againto FIG. 1, the attenuation time of the emitted light (i.e. thefluorescence lifetime, representing the average amount of time amolecule remains in the excited state prior to returning to a groundstate) is determined based on the intensity of the fluorescenceemission, using the output of streak camera 9 on a cell-by-cell basis.The output from camera 9 and a peak threshold value 12a are input tocomparator 13, and, when a decreasing light emission signal reaches thepeak threshold value, a counter 15 is started and continues to countuntil the light emission signal reaches an attenuation threshold value14 (typically 1/e or 63% of peak) when the count is terminated. Theprocessor 10 uses the attenuation time count for each cell to perform ananalysis of the sample cell population and even control cell sortingwith sorter 11.

However, this streak camera approach encounters significant difficultiesdue to the need for highly accurate and expensive counters. Also, thisapproach is dependent on the intensity of the emitted light, which willrequire the peak threshold to be varied. Moreover, if variable signalattenuation is present, due to the dependence of the count on emittedlight intensity, the measuring of attenuation time may not beconsistent. Furthermore, the use of a synchro-scan streak camera has thedisadvantage of high cost, high complexity and limited sensitivity,owing to its extremely small sensitive area.

In a similar intensity-based system disclosed in U.S. Pat. No.4,778,539, Yamashita et al, issued Oct. 18, 1988, individual cells maybe distinguished by measuring light at an observation point of a flowcytometer system. There, the change in the intensity of transientemitted light over a period of time, following excitation by shortpulses of laser light, may be measured and used to detect theattenuation time of the emitted light, the rise time of the emittedlight and the orientation relaxation time. These parameters may be usedas a basis for cell discrimination. However, such measurements areintensity-based and are performed cell-by-cell, and do not allow forrapid and/or simultaneous scanning of a population of cells, based onlifetime measurements.

A suggestion that phase based measurement of fluorescence lifetimes maybe employed in flow cytometric systems appears in Cytometry, Supplement2, p. 91 (1988), Steincamp et al. However, there is no specificdisclosure of the structure or method of operation of a system which canperform the identified function.

Thus far, practitioners have been unsuccessful in measuring fluorescencelifetimes with phase-based techniques and in using such measurements toidentify the existence of particular analytes without complicatedprocessing of the detected data signals. Indeed, where such signalprocessing is used to determine the lifetime of radiated cells within aflow cytometer, the performance of a selective sorting of such cells hasbeen difficult since the processing may not be sufficiently rapid toenable sorting of the cells as they flow through the cytometerobservation point.

In a system not previously used for flow cytometry, phase-modulationfluorometry and phase-sensitive fluorescence spectroscopy (PSFS) providea means by which fluorescence lifetimes of one or a few fluorophores inhomogeneous solutions are measured for the study of specific fluorophorelifetimes. One approach, described in PRINCIPLES OF FLUORESCENCESPECTROSCOPY, J. R. Lakowicz, Plenum Press (1983), discloses a techniquein which a sample containing only one or a few fluorophores is excitedwith light having a time-dependent intensity and a detection is made ofthe resulting time-dependent emission. Because the emission isdemodulated and phase shifted to an extent determined by thefluorescence lifetime of the species, the fluorescence lifetime (τ) canbe calculated from the phase shift O of the species: ##EQU1##

or from a demodulation factor m: ##EQU2## where m is a demodulationfactor, ω is the angular modulation frequency and O is the phase shiftof the species. Phase sensitive detection results from a comparison ofthe detected emission with an internal, electronic reference signal ofthe same frequency.

In a second approach, described in "Phase-Sensitive FluorescenceSpectroscopy: A New Method To Resolve Fluorescence Lifetimes Or EmissionSpectra Of Components In A Mixture Of Fluorophores" Lakowicz et al,Journal of Biochemical and Biophysical Methods; vol. 5, 1981, pp. 19-35,the time-dependent fluorescence photodetector output signal ismultiplied by a periodic square-wave signal which has the samemodulation frequency as the fluorescence signal, and is then integrated.A time-independent dc signal is thereby produced that is proportional tothe cosine of the difference between the phase angles of the square-waveand the fluorescence signal, and proportional to both signal amplitudes.

The two approaches described above are related to periodically modulatedfluorescence excitation light. Consequently, continuous output signalsare generated in both techniques. Therefore, these standard techniqueswould not have been considered to be used to measure fluorescencelifetimes of single cells passing the observation region in a flowcytometer. Moreover, as already mentioned, the output signal in thesecond technique depends not only on the fluorescence lifetime but alsoon the fluorescence signal intensity. As a matter of experience, thefluorescence signal intensity observed in a flow cytometer variessignificantly from cell to cell, and therefore phase-modulationfluorometry and PSFS would not have been considered to be useful forflow cytometric measurements. Also, known phase modulation fluorometrymethods use slowly responding circuits which would be expected toprevent cell-to-cell measurements.

SUMMARY OF THE INVENTION

According to the present invention, there is provided an apparatus andmethod for determining, in a flow cytometric environment, valuescorresponding to lifetimes of fluorescence emitted by single cells orparticles associated with one or more fluorophores, in response to theillumination of the cells by modulated light of a wavelengthcorresponding to the excitation wavelength of the fluorophore.

Such a method of the present invention comprises directing a stream ofindividual cells past an observation point, irradiating each cell in thestream of cells with light modulated by an input signal at a presetfrequency, and detecting the fluorescence emission of the fluorophore toproduce a corresponding emission signal. There is generated an outputsignal which represents the change in phase and/or modulation betweenthe input signal and the emission signal, which is indicative of thefluorescence lifetime of the fluorophore, and which is independent ofthe intensity of emitted light from the excited fluorophore.

One object of the present invention is to provide a method and anapparatus which increase the measurement, resolution, analysis andsorting capabilities of flow cytometers.

Another object of the present invention is to perform fluorescencelifetime measurements on single cells using phase resolution based onthe decay time measured by the phase angle which is independent of theintensity of the emitted fluoresence.

Another object of the present invention is to implement a fluorescencelifetime measurement system which has an accurate and rapid response andis not dependent upon the intensity of the measured fluorescence.

Another object of the present invention is to implement a phase angle ormodulation based fluorescence lifetime measurement system forapplication to a flow cytometer, which is simple, low cost and requiresno counting circuitry.

In one embodiment, the detecting step is performed by means of a PMT,and the output signal is generated as a ratio of the the split PMTsignals. The PMT signal is split, one of the signals is phase shifted90°, and the two signals are ratioed to create an intensity-independentamplitude which reveals the phase angle. In another embodiment the PMTsignal is mixed with a variable-phase input signal, and the mixer outputis integrated and used to control the input signal to provide to theintegrator a voltage output that corresponds to the phase shift of theemission signal relative to the phase of the input signal.

In another embodiment, in the irradiating step, the light source is async-pumped and cavity-dumped dye laser which is intensity modulated bya mode-locked signal, corresponding to the input signal, via a frequencysynthesizer. According to another aspect of the present invention, aflow cytometer is provided that is operative to measure a fluorescencelifetime of at least one fluorophore associated with a cell or aparticle, comprising: a flow chamber for directing a plurality of thecells or particles past an observation point; a modulated light sourcefor irradiating, at the observation point, each cell or particle withmodulated light at a frequency of an input signal and having awavelength capable of exciting the fluorophore to produce an emission; aphotodetector for detecting the emission and producing a correspondingemission signal; and a phase/modulation detector for generating anoutput signal corresponding to a change in phase and/or modulationbetween the emission signal and the input signal, wherein the outputsignal is indicative of the fluorescence lifetime of the fluorophore andis independent of fluorescence intensity.

In one embodiment, the photodetector is a PMT, and the apparatus furthercomprises a mixer and an integrator for mixing and integrating theoutput signal and the input signal to produce the voltage signal. Inanother embodiment, the integrator produces a signal having a constantvoltage, wherein the voltage corresponds to the phase shift of themodulated fluorescence radiation.

In another embodiment, the light source is a sync-pumped andcavity-dumped dye laser which is intensity modulated by a mode-lockedsignal, corresponding to the input signal, via a frequency synthesizer.

According to another aspect of the present invention, there also isprovided an apparatus for determining the lifetime of at least onefluorophore that is associated with a cell or particle in a flowcytometer, comprising a source of high intensity light at a selectedwavelength capable of exciting the at least one fluorophore andmodulated by an input signal at a set frequency, the modulated lightbeing directed onto individual cells or particles, associated with theat least one fluorophore, passing an observation point in the flowcytometer to excite the fluorophore, a detector for detecting theemission from the excited fluorophore at the observation point togenerate an emission signal, circuitry for splitting the modulatinginput signal into first and second signals that are in phase quadratureand for splitting the emission signal into third and fourth signals,circuits for mixing each of the first and second modulating signals witha respective one of the third and fourth emission signals, and ratiocircuitry for creating the ratio of each of the two pairs of mixedsignals to provide a lifetime signal which corresponds to the lifetimeof the at least one fluorophore, such that the lifetime measurement isindependent of the intensity of the fluorescence emission from theexcited fluorophore.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a conventional flow cytometric system whichcan detect fluorescence lifetimes;

FIG. 2 is an illustration of a first embodiment of the presentinvention.

FIGS. 3A-3G show waveforms of signals in the embodiment shown in FIG. 2.

FIG. 4 is a schematic block diagram of a second embodiment of thepresent invention.

FIGS. 5A and 5B are schematic block diagrams of a third embodiment and avariation thereof, respectively.

FIG. 6 is a schematic block diagram of a fourth embodiment of thepresent invention.

FIGS. 7A-7F are graphical representations of waveforms produced in phasefluorescence lifetime measurements.

FIG. 8 is a graphical representation of phase fluorescence versusfluorescence amplitude of cell nuclei stained with ethidium bromide andfluorophore dyed particles.

FIG. 9 is a graphical representation of phase fluorescence versusfluorescence amplitude of cell nuclei stained with ethidium bromide andfluorophore dyed particles with the use of an optical band pass filter.

FIG. 10 is a graphical representation of phase fluorescence versusfluorescence amplitude of cell nuclei stained with ethidium bromide andfluorophore dyed particles with the use of an optical band pass filterwith the PMT voltage held constant.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The method and apparatus of the present invention will now be describedin detail with reference to the accompanying drawing figures.

In contrast to the flow cytometry using intensity and/or wavelengthbased fluorescence detection, the present invention was discovered tounexpectedly provide apparatus and methods for characterizing and/ordistinguishing cells or particles based on an average variation in thelifetimes of most or substantially all of the fluorophore moleculesassociated with cells or particles in a sample, which variation isdirectly due to the presence of an analyte in the cell or particle. Suchvariations have now been discovered to be dependent on the amount,concentration, or physical or chemical state of the fluorophore beingsensed, such that practicable and commercially useful cell or particlecharacterization or distinguishing methods and apparatus can be providedaccording to the present invention.

Accordingly, analyte sensing can be achieved by methods of the presentinvention in which fluorophores, having emissions with a known lifetimewhen stimulated by light at a predetermined wavelength, are excited by alight source, such as a high frequency light source.

In the context of the present invention, the term "sample", whichcontains cells or particles to be used in flow cytometry according tothe present invention, refers to compounds, mixtures, surfaces,solutions, emulsions, suspensions, mixtures, cell cultures, fermentationcultures, cells, tissues, secretions and/or derivatives or extractsthereof. Samples, as defined above, which can be used in methods of thepresent invention for sensing analytes based on fluorescence lifetimesalso include samples that can be clear or turbid. Such samples to bemeasured according to the present invention require only that thefluorophore used be contacted with the sample such that the analyte tobe sensed influences the lifetime of the fluorophore such that thelifetime varies with the presence or amount of the fluorophore.

Such samples can also include, e.g., animal tissues, such as blood,lymph, cerebral spinal fluid (CNS), bone marrow, gastrointestinalcontents, and portions, cells or internal and external secretions ofskin, heart, lung and respiratory system, liver, spleen, kidney,pancreas, gall bladder, gastrointestinal tract, smooth, skeletal orcardiac muscle, circulatory system, reproductive organs, auditorysystem, the autonomic and central nervous system, and extracts or cellcultures thereof. Such samples can be measured using methods of thepresent invention in vitro, in vivo and in situ.

Additionally, samples that can be used in methods of the presentinvention include cell culture and fermentation media used for growth ofprokaryotic or eukaryotic cells and/or tissues, such as bacteria, yeast,mammalian cells and insect cells.

The term "analyte" in the context of the present invention refers toelements, ions, compounds, or salts, dissociation products, polymers,aggregates or derivatives thereof.

FIG. 2 is a block diagram of the overall configuration of an apparatusfor phase-angle detection of the fluorescence lifetime of a cell orparticle sample in a flow cytometer. Elements from the conventional flowcytometer system of FIG. 1 are identified by the same reference numberswithin the flow cytometric environment of FIG. 2. Cell samples would bedyed with a suitable fluorescent dye having a known absorptioncharacteristic for light within a particular wavelength and emittinglight at a particular wavelength. Examples of such dyes are given inTable 1.

                  TABLE 1    ______________________________________                Laser      Dye                Utilized for                           Excitation/                                      Dye                Dye Excita-                           Absorption Emission    Dye Type    tion       Wavelength Wavelength    ______________________________________    DNA-staining dyes                Argon,     325-355 nm 450 nm (blue)    Hoechst 33342/                Helium-    (UV)    DAPI        Cadmium    Mithramycin Argon      457 nm     575 nm                           (blue-violet)                                      (green)    Chromomycin Argon      457 nm     555 nm                           (blue-violet)                                      (green)    Propidium iodide                Argon      342-514 nm 615 nm                           (UV to     (orange-red)                           yellow)    RNA-staining dyes                Argon      480-550 nm 570-600 nm    Pyronin Y              (blue-green)                                      (orange-red)    Protein-staining                Argon      488 nm     520 nm    dyes                   (blue-green)                                      (green)    Fluorescein    Phycoerythrin                Argon      488 nm     578 nm                           (blue-green)                                      (orange-red)                           peak 565 nm)    Texas Red   Krypton    590 nm     615 nm                           (green-    (orange-red)                           orange)    Allophycocyanin                Krypton/   635-650 nm 670 nm                Helium-Neon                           (red)      (deep red)    ______________________________________

Many other compounds display changes in fluorescent lifetime in responseto a variety of environmental parameters such as temperature, DNAcomposition, the presence or concentration of elements such as oxygen,sodium ions, calcium ions, potassium ions, magnesium ions and theexistence of certain pH levels. For example, calcium ions in a cell orparticle could be measured according to the present invention using anArgon laser and the flourophore Calcium Green. As would be clear to oneof ordinary skill, currently known analytes, and those which may bedeveloped in the future, having a known sensitivity to light at aparticular wavelength, may be used.

Fluorophores suitable for use in methods or apparatus according to thepresent invention include fluorophores whose lifetimes vary directlywith a concentration or amount of a particular analyte, as describedherein, and which lifetimes are detectable by known fluorescencespectroscopy methods.

Accordingly, analyte sensitive fluorophores suitable for use in methodsof the present invention include fluorophores that have fluorescentlifetimes which vary continuously over a suitable range of analyteconcentrations or amounts, and are excitable with a suitable chromaticlight, such as a laser light at a corresponding wavelength. Thus, thepresent invention provides for flow cytometry including the use of allfluorophores meeting the above criteria. These criteria can bedetermined by routine experimentation using known procedures which willbe apparent to one of ordinary skill in the art. Therefore, fluorophoressuitable for use in methods and apparatus of the present invention arenot limited to the types and examples described herein, and are nowdiscovered to correspond to those fluorophores which can be used forwavelength and intensity measurements. Fluorphores suitable for use inapparatus or methods of the present invention are available, e.g., fromMolecular Probes, Inc., Junction City, Oreg.

In operation, as shown in FIG. 2, samples having a dye representative ofthe analyte to be detected are present in solution and are directed fromthe sample source 4 into the sample chamber 5. The sheath fluid fromsource 6 aligns the cells and causes them to pass in a stream, one cellat a time, past an observation point in the flow chamber 3 of the flowcytometer, in a manner well known in the art. At the observation pointeach cell in the stream of cells is irradiated, and the resultingradiation emission from each cell is detected. After passing through theobservation point, the stream of cells may be formed into uniformdroplets and sorted by a cell sorter 11, if desired. In accordance withthe present invention, as further described herein, the sorting may beaccomplished on the basis of detected fluorescence emission signals orthe derived lifetime values for each cell.

Light source 20 is a periodically pulsed laser, whose output is directedto the source projection port 3a of flow chamber 3 via an excitationoptics subsystem 2. In its preferred form, the light source is amode-locked laser which is capable of generating pulses of light,exhibiting a broad spectrum of wavelengths in the ultraviolet range, ata fast repetition rate. The mode-locked laser is self triggering andrequires no external timing, yet it can produce pulses at a repetitionfrequency in the Mhz-range which is sufficient to illuminate each cellwith a pulse of light at least one time as it passes through theobservation point of flow chamber 3. A preferred light source wouldcomprise an argon laser, an internal modulated helium-Neon (HeNe) laseror a frequency-doubled YAG laser having a mechanism for generating lightpulses by mode locking, a frequency-doubled dye laser for performing awavelength conversion which permits the laser wavelength to match thesensitivity of the probe, and a cavity dumper for controlling thegeneration of light pulses at a desired rate and width. An appropriatepulse rate for the preferred embodiment would be≧4 MHz. Yet anotherlight source structure may be found in the flow cytometer manufacturedby Becton Dickinson Corporation, and sold under the trademark FACScan,where an air-cooled low-power 15 mW argon-ion laser operating at 488 nm,is used. The continuous light generated by the argon-ion laser would beexternally modulated by an acousto-optical or electro-optical modulatorto produce the desired pulsed or sinusoidal output, as will be explainedin greater detail herein.

Light sources suitable for use in the methods of the present invention,also include noble gas light sources such as neon and argon andcombinations, thereof. Light sources can include gas lamps or laserswhich provide uniform light that has been filtered, polarized, orprovided as a laser source, such as a coherent wave (CW) laser or adiode laser, e.g., a pulsed dye laser. Specified impurities can be addedto the above described noble gas light sources to provide suitable lightsources for use in the present invention with varying wavelengths suchas emission and excitation wavelengths. Such impurities include Group IImetals, such as zinc and cadmium. For example, a green helium neon laseras the light source for measuring fluorescent lifetimes according tomethods of the present invention.

The light source can be modulated by the optic modulator as anacousto-optic modulator or an electro-optic modulator. Alternatively,the laser can be a periodically pulsed dye laser producing a laser pulserepetition frequency.

The beam emitted by the periodically pulsed laser 20 may beappropriately shaped by an excitation optics subsystem 2. For example,the Becton Dickinson FACscan cytometer employs a prismatic expander andspherical lens to provide an elliptical beam input to the sourceprojection port 3a of a flow chamber. As each of one or morefluorophores that are associated with a cell or particle passes throughthe observation point in the flow chamber, the fluorophore will beexcited by one or more pulses of light. A fluorescence emission of thefluorophore associated with the cell or particle as a result ofillumination by the laser beam will result when the wavelength of thelaser light matches the absorption band of one or more fluorophoresused. The fluorescence emission will be at a longer wavelength and willhave a delay time that characteristically identifies the fluorescentspecies, and can be modified by the presence of an analyte in orassociated with the cell or particle.

In the present invention, such a fluorescence emission is received by anemission optics subsystem 8 which is designed to collect, direct andfocus the light emitted at the flow chamber observation port 3b, as thesample passes through the observation point. Typically, such emissionoptics comprise an emission lens coupled to the flow chamber observationport 3b with the refractive indices matched by an optical gel. The lightcollection system may comprise a plurality of dichroic mirrors, whichpass light at predetermined wavelengths and reflect light at otherwavelengths. The optical system may use filters to separate the emittedlight into its primary colors, and direct each monochromatic light beaminto a separate channel for detection and analysis. In the preferredembodiment, plural channels are contemplated; however, for ease ofillustration, only a single channel is shown in FIG. 2. It should beunderstood by one of ordinary skill that a plurality of channels, onefor each respective monochromatic beam of light, would be employed. Theemitted light at each color would be detected by an optical transducer,such as a photodetector 29.

The photodetector 29 is a sensitive, high speed device such as a ModelR2496 or R928, manufactured by Hamamatsu Photonics K.K. Thephotodetector generates an output having a duration and amplitude whichdepends upon the duration and intensity of the received light beam atthe wavelength to which the photodetector is sensitive. Thephotodetector produces a pulsed current output voltage which, inconventional flow cytometry systems, are directed to a signal processingsubsystem for fluorescence analysis.

In accordance with the present invention, however, the laser lightsource 20 is itself subject to pulse or sine wave amplitude modulation.This modulation may be internally generated for a mode-locked laser ormay be externally generated by a trigger circuit, as in the case of acavity dumped laser, or a local frequency generator generating a sinewave at a desired modulation frequency. The schematic illustration ofFIG. 2 illustrates a separate modulator driver 21, but is intended torepresent an internally generated modulation as well. This unit providesfrom the laser a sinusoidal output. In the preferred embodiment, themodulating frequency would be within the GHz to MHz-range, e.g., at 80MHz. The laser modulator may be a TEM-20-7.5 Model modulatormanufactured by Brimrose Corp. of America, and having an auxiliarysynchronization output. When the laser is mode-locked and generates themodulation internally, the modulation frequency can be detected at apoint on the laser. The exciting laser beam, whether mode-lockedinternally or modulated externally, has a periodically varyingintensity.

The intensity of the fluorescence emitted by the excited probe materialis directly dependent on the intensity of the excitation beam. Thus, thefluorescence emission will be modulated at the same frequency as theexciting beam. In the preferred embodiment, since the exciting beam ismodulated at a 80 MHz frequency, the fluorescence response will exhibitan intensity which also varies at a 80 MHz frequency. Recognizing that agraphical representation of the pulsed fluorescence signals will be forillustration only, a representation of a sinusoidal excitation outputintensity I₁ from the laser 1 unit is seen in the waveform illustratedin FIG. 3A. This waveform varies at a frequency f1 which is 80 MHz inthe preferred embodiment. A representation of the intensity I₂ of thefluorescence emitted from the sample, following illumination by themodulated light source, is seen in FIG. 3B. From the waveformillustrated in that Figure, it is clear that the emission is modulatedat the same frequency f1 as the exciting beam and has a sinusoidalcharacteristic but experiences a phase lag (P) relative to the excitingbeam. The phase lag P results from the characteristic delay which isexperienced by a fluorophore in emitting its fluorescence afterexcitation. The phase lag P is seen by a comparison of the waveformsillustrated in FIGS. 3A and 3B. It has been determined that the phaselag P is related to the fluorescence lifetime τ in accordance with thefollowing equation:

    tan(P)=2πfτ,

where f=the modulation frequency.

The structure of the invention as illustrated in FIG. 2 is intended toutilize this relationship and to provide an output signal with anamplitude that is proportional or otherwise related to the fluorescencelifetime. This relationship may be established empirically, or therelationship may be understood from the known nature of intensity decayand the phase and/or modulation detection circuits. Referring again toFIG. 2, the output from the modulator driver 21 and the output detectedby the photodetector 29 are processed in order to derive anintensity-independent signal which is indicative of fluorescencelifetime. An output is taken from the auxiliary terminal of modulatordriver 21 (or directly from a terminal of a mode-locked laser) and isdirected via variable phase shifter 22 to a two-way 0° power splitter23. The signal output of the photodetector 29 also is connected to theinput of a two-way 0° power splitter 24. Each of the two power splitterscan divide the power of the input signal in a specified proportionbetween each of its respective two output ports, without affecting thefrequency and phase of the input signal. In the preferred embodiment,the power division is equal; each output of power splitter 23 isone-half of the power input from variable phase shifter 22 and retainsthe same modulation frequency and phase of the signal generated at theoutput of the modulator driver 21. In this first embodiment, variablephase shifter 22 is assumed to provide no phase shift in the output frommodulator driver 21; however, as disclosed subsequently, a specifiedshift may take place in order to suppress the unwanted signals of autofluorescence and/or stray light.

Each of the two RF outputs from power splitter 24 also receives one-halfof the power of the output signal from photodetector 29 and maintainsthe frequency and phase of that output signal. Thus, the two RF outputsfrom power splitter 24 have a 80 MHz frequency and have a delayed phaserelationship with respect to the RF outputs of the power splitter 23.

The two outputs of power splitter 24 are connected to the RF inputs of afirst phase detector 25 and a second phase detector 26. The waveforms ofthese signals correspond to the fluorescence waveforms shown in FIG. 3B.These two phase detectors are RF mixers with a low DC offset and with ahigh figure-of-merit, measured in maximum DC output voltage in mVdivided by the RF input power in dBm. The respective outputs of each ofthe first phase detector 25 and second phase detector 26 are provided toa ratio unit 27 which generates an output signal corresponding to theratio of the output signals of the phase detectors 26 and 25. Such ratiounit may be a Model AD 539 manufactured by Analog Devices.

A second input signal to the first phase detector 25 is provided by oneoutput 23a of power splitter 23. This RF signal is shown in FIG. 3C. Aspreviously noted, the first phase detector 25 is an RF mixer whichreceives the output 23a of the power splitter 23 at its LO input andproduces a first input B to the ratio detector 27. The signal B is shownin FIG. 3D. In operation, the output of the phase detector 25 is an IFsignal that is proportional to I cosD, where I is the fluorescenceintensity and D is the phase difference between the photodetector RFsignal and the synchronization output LO signal.

The other output 23b of power splitter 23 is connected via a fixed90-degree phase shifter 28 to the LO input of the second phase detector26. The phase-shifted RF signal is shown in FIG. 3E. Due to the fixed90-degree phase shift, the output IF signal A of the phase detector 26,illustrated in FIG. 3F, is proportional to I sinD, where I and D havethe same meaning previously discussed with respect to the output ofphase detector 25.

The ratio unit 27 receives a signal I cosD and I sinD and generates anoutput signal R which corresponds to the ratio between the outputsignals of the phase detectors 26 and 25. Referring to the value ofthese output signals, the ratio unit output amplitude is represented bythe following equation:

    R=(I sinD)/(I cosD)=tanD.

The amplitude of the output from the ratio unit 27 is independent of thefluorescence intensity I, as is indicated in FIG. 3G. For fluorescenceradiation, the output intensity is R=tanD=2π.f.T where f is the LOfrequency which is constant for a particular measurement, and τ is thefluorescence lifetime. The ratio unit output amplitude R, is thusdirectly related to the fluorescence lifetime τ and is independent ofthe fluorescence intensity I. In practice, the signal may not beprecisely proportional to the lifetime but, in any event, will besufficiently related to lifetime so as to enable discrimination amongparticles.

The output amplitude R may be detected and processed by conventionalhardware and software 30, currently capable of processing the pulseamplitude output from existing flow cytometers. A computer can convertthe output R into decay times and use such decay times for analysis andcell sorting. Thus, using existing fluorescent probes, it is possible tomeasure cellular pH, cations, oxygen concentration and Na⁺ compositionsince fluorophores are known whose decay times are sensitive to theseparameters. With the advent of this new technology, fluorophores will bedeveloped whose decay times are sensitive to a variety of analytes andwill allow enhanced study of cellular physiology on a cell-by-cellbasis. For example, fluorescence lifetimes may depend on theconcentration of particular analytes. Therefore, the measurement of suchlifetimes can identify the presence of those analytes. Fluorescentcompounds, which display changes in lifetimes in response totemperature, oxygen, pH, and the presence of particular elemental ionssuch as those of sodium and calcium, may be used.

The flow cytometer with phase fluorescence lifetime measurement ability,as seen in FIG. 2, also has an autofluorescence signal suppressioncapability. The light detection system may be adjusted electrically insuch a way that the output signals of unwanted autofluorescence and/ornon-rejected stray light become equal to zero. As seen in FIG. 2, thevariable phase shifter 22, which receives the output of modulator driver21 (or the detected output of a mode-locked laser) and provides thatmodulation signal to power splitter 23, may be set to provide a zerophase shift in a first embodiment. However, in a second embodiment, thesignal contribution of unwanted autofluorescence can be suppressed ifthe variable phase shifter 22 is adjusted so that the phase differencefor autofluorescence radiation at phase detector 26 is equal to zero.Alternatively, the phase of the LO input to 25 is adjusted to be 90°shifted from the input signal to 25. By reducing the autofluorescencesignal to zero, fluorescence with a lifetime different from theautofluorescence lifetime can be easily detected. When A is the phaseshift angle resulting from the autofluorescence lifetime τ_(A), thevariable phase shifter 22 has to be adjusted to a shift angle equal toA. Under these conditions the output amplitude of the ratio device forautofluorescence present may be given by the expression:

    R= sin (D-A)!/ cos (D-A)!=tan (D-A).

Thus, fluorescence with a lifetime T longer than τ_(A) generatespositive output amplitudes, auto fluorescence with a lifetime τ=τ_(A)generates no signal, and fluorescence with lifetimes τless than τ_(A)generates negative amplitudes. As in the first embodiment, theamplitudes in this embodiment are independent of the fluorescenceintensity and can easily be corrected by a computer in order tonormalize the output signal to only positive values.

The signal-suppression capabilities of a flow cytometer according to thepresent invention are not limited to autofluorescence with asingle-exponential decay. For an autofluorescence showing any kind ofkinetics, there exists a specific phase setting of the variable phaseshifter that causes the autofluorescence signal to become equal to zero.This principal is applicable also to a combination of autofluorescentlight and unrejected scattered radiation. The only condition that mustbe fulfilled in this case is that the relative contributions ofautofluorescence and scatter must remain constant.

FIG. 6 shows a further embodiment of the present invention which allowsfor phase detection without intensity dependence and which involvesmodifications to the embodiment, as variations thereof, presented inFIG. 2.

In particular, in FIG. 6, the split output signal 24a of the powerspitter 24 (FIG. 2) is fed via the high pass filter 202 to an additionalnon-phase shifting power splitter 201 which provides two split outputsignals 201a and 201b which are routed through respective limiters 215aand 215b after amplification in respective variable gain amplifiers 213and 214. The modulating signal from the frequency generator 21 and thephase-shifter 22 shown in FIG. 2 is also split by a power splitter 23 intwo equal output signals 23a and 23b.

The split output signal 23b is phased shifted by 90 degrees in a phaseshifter 28, while the other split output signal 23a is kept in phasewith the output of the frequency generator 21 (FIG. 2). The in-phase andphase-shifted signals are amplified by respective amplifiers 219a and219b, limited by respective limiters 220a and 220b and fed to respectivedouble balanced mixers 203a and 203b where they are mixed with the splitand limited fluorescence output signals 201a and 201b, respectively. Theresulting output signals of the mixers 203a and 203b are low passfiltered via low pass filters 204a and 204b and peak detected via peakdetectors 205a and 205b, which are gated by the comparator circuit 207to produce two output voltages 206a and 206b corresponding to cosine andsine, respectively of the emission signal of the detector PMT 29 of FIG.2. The gating is accomplished via the comparator 207 which comprises apreamplifiers 207a and a comparator 207b whose output signal gates orenables the peak detectors only after the envelope of each peak haspassed a predetermined threshold value, determined by a reference signalinputted by the comparator 207b as a predetermined threshold levelcorresponding to amplitude limits encompassing the amplified and limitedfluorescence output of the detector PMT 29 (FIG. 2), and disables thedetectors 205a and 205b when the signal envelope drops below thethreshold. The ratio of the output voltages 206a and 206b is thendetermined by a modulation ratio unit 221 whose output is intensityindependent, since the peak voltage is representative of the tangent ofthe phase and/or modulation change between the fluorescence outputsignal originating from the detector PMT 29 and the reference signalfrom the frequency generator 22 (FIGS. 2). This change in phase and/ormodulation corresponds to the fluorescence lifetime of the fluorophoreassociated with the cell or particle passing through the flow cellchamber 3 (FIG. 2). To determine this change in phase and/or modulation,the output voltage from the modulation ratio unit 221 is fed into thesignal processor 30.

Optionally, a limiting circuit can receive and limit the output signalproduced by the photodetector. Limiting circuits are described, e.g.,Spencer, thesis, University of Illinois at Urbana-Champaign, 1970. Apreferred limiting circuit is model PLS-1 from Mini-Circuits, Brooklyn,N.J. The limiting amplifier results in phase angle measurements based ondetection of zero crossing, which are mostly independent of signalamplitude. While the limiting circuit produces additional harmonics ofthe signal from the photodetector, the effect of the limiting circuit isto eliminate all signals above a preset level. The limiting circuitprovides such a centroid signal which maintains the same phasecharacteristics of the output signal which the output signal had priorto comparison of the output signal with the input signal of the lightsource.

While some phase shifting of the signal can occur with the use of alimiting circuit, the harmonics and band noise produced by the limitingcircuit can be removed by the use of one or more known low and high passfilters. Preferably, the output signal from the photodetector can be lowand high pass filtered and amplified with an optional filter/amplifier,prior to limiting by the limiting circuit.

Accordingly, an alternative embodiment of the present invention is shownin FIG. 5A. A laser source 100 produces a laser beam which is frequencymodulated at a frequency of up to 100 MHz with an acoustic opticmodulator 101 driven by a frequency generator 111 to produce a modulatedlaser light beam 101a. The modulated laser light beam 101a passesthrough a beam splitter 120a and illuminates a fluorophore-associatedcell, bead or particle in a flow cell 119 to produce a fluorescenceemission from the fluorophore. The resulting fluorescence emission isthen detected by a detector 102 at a side scattered position of 90 to175 degrees to the incident modulated laser beam 101a. The detector suchas a PMT 102, detects the fluorescence emission and produces acorresponding electrical output signal.

The fluorescence electrical output signal, after 10 dB of amplificationby a preamplifier 103, is split into two equal signals 104a and 104b,respectively, by a power splitter 104 while maintaining fidelity andrelative amplitude without inducing additional intensity dependent phaseshifts. A first signal 104a from the power splitter 104 is fed through ahigh pass filter 105 and then mixed and further amplified in a variablegain amplifier 106, whose output 106a is split by a power splitter 112to provide a first split signal 112a which limited to a suitableoperating range by a limiting circuit 107. The second split signal 112bis peak detected in a peak detector 114c forming part of a gated peakdetector circuit 114.

The output 107a of the limiting circuit 107, as a limited signal whichphase shifted relative to a reference signal (REFa or REFb, as presentedbelow), is then mixed (e.g., multiplied) via a double balanced mixer 108with the reference signal REFa or REFb obtained as follows:

(A) A reference signal REFa (FIG. 5A) is produced in the followingmanner. A detector, such as a PMT 109, detects the modulated laser lightbeam 101a which has been split by a beam splitter 120a and reflected bya mirror 120b. The detector PMT 109 then produces a corresponding outputelectrical signal which is amplified by 10 dB by a preamplifier 130,amplified by a variable gain amplifier 117 and then limited by alimiting circuit 118 to produce the reference signal REFa.

(B) A reference signal REFb (FIG. 5B) is produced in the followingmanner. The electrical output signal from the frequency generator 111 issplit by a power splitter 115 into two signals 115a and 115b,respectively. The signal 115b drives the acousto-optic modulator 101.The signal 115a is amplified by a variable gain amplifier 117 and thenlimited by a limiting circuit 118 to produce the reference signal REFa.

The multiplied output signal of the mixer 108 is produced by the mixingof (1) the reference signal REFa or REFb from the limiter 118 and (2)the phase shifted limited signal 107a from the limiting circuit 107. Themultiplied output signal from the mixer 108 is filtered via a low passfilter 110 and the peak voltage of the multiplied output signal is ameasure of the relative phase shift of the output signal from the PMT102 to the reference signal REFa or REFb originating from the frequencygenerator 111. The filtered multiplied output signal from the mixer 108via the low pass filter 110 is detected via a peak and hold circuit inthe flow cytometer 140. The fluorescent lifetime is related to thedetected output signal as follows:

    Vp=v+c* cos (phase)

where Vp=peak voltage

c is proportional to pulse amplitude,

v is an offset voltage unique to the limiter, and

phase=arc cos ((Vp-v/c); and

T=(tan(phase)/2*π*f, where π=3.1416, f is operating frequency and τ isthe fluorescence lifetime.

The signal 104b obtained from the power splitter 104 is filtered by alow pass filter 113 to produce a low pass filtered signal detected via agated peak detector circuit 114 comprising a comparator 114a and a peakdetectors 114b and 114c. The amplitude of the low pass filtered signalis gated to the peak centroid via the comparator with a referencesignal, inputted as a predetermined threshold level corresponding toamplitude limits encompassing the amplified and limited fluorescenceoutput of the detector PMT 102, and a peak detector 114b to produce anoutput signal B. The high pass filtered signal, originating from thepower splitter 104 as signal 104a, is gated via a peak detector 114c toproduce a signal A. The gated high pass filtered signal A is thencompared to the gated low pass filtered signal B and a modulation ratiois determined via a modulation ratio unit 116.

This modulation ratio is then inputted into the flow cytometer 120 andprovides a measure of the relative depth of modulation "M" between the(1) the reference signal REFa or REFb from the limiter 118 and (2) thephase shifted limited signal 107a from the limiting circuit 107.

When the flow cytometer determines the lifetime of the fluorophore basedon the modulation ratio, if the first order of the reference signal REFb(FIG. 5B) is used to determine the depth of modulation "M", then themodulation ratio will directly correspond to the depth of modulation andno correction factor will be needed. However, if the zeroth order of thereference signal REFb (FIG. 5B) is used to determine the depth ofmodulation "M" then the modulation ratio will not directly correspond tothe depth of modulation and a correction factor of percent modulation ofthe reference signal will be necessary.

The ratio modulation outputted from the modulation ratio unit 116 andmultiplied output signal from the mixer 108 via the low pass filter 110are inputted into the flow cytometer 130 to provide the apparentfluorescent lifetime based on (1) the modulation ratio of the peakvoltages from the peak detectors 114b and 114c and (2) change in phaseof the detector 102 output signal, as compared to the reference signalREFa or REFb.

The resulting peak voltage is a measure of the apparent fluorescentlifetime by the following relationship.

    t=(square root of((1/(M*M)).sup.-1)/(2*π*f)

where M=the ratio of the amplitudes.

An alternative embodiment of an apparatus and method according to thepresent invention is shown schematically in FIG. 4. The output light ofa laser 301 is intensity modulated by a optical modulator 302 driven bya sinusoidal modulation signal from an electrical modulation driver 303.The resulting intensity modulated laser light is deflected via adeflector 304 and a deflection driver 304(a) onto a sample 305containing one or more analytes, particles or cells. A fluorescencephotodetector, such as a PMT (photomultiplier tube) 306, detects thefluorescence emission and produces an electrical output signal which isfed to the RF input of a mixer 308. The sinusoidal modulation signalfrom the modulation driver 303 is also fed to the input of a voltagevariable phase shifter 307 whose output is connected to the LO input ofthe mixer 308, and the IF output of the mixer 308 is fed to anintegrator 309, whose output is fed to the voltage-control input of thevariable phase shifter 307. The integrator output contains the phasechange or modulation information, thereby providing the cell's change inlifetime due to the presence of the analyte, particle or cell associatedwith the fluorophore.

When the RF signal and the LO signal at the mixer are 90° out of phase,the IF output signal is equal to 0. When the fluorescence light isphase-shifted by an angle θ relative to the laser light source, themixer IF output signal is different from 0. This IF signal isintegrated, and the voltage of the integrated output signal is fed backto shift the LO signal phase the required amount to place the RF signaland the LO signal 90° out of phase at the mixer 308. When this conditionis achieved, the IF output signal of the mixer 308 again becomes equalto 0, and the integrator 309 output voltage remains constant. Since thisrequired LO signal phase shift is identical to the fluorescence phaseshift angle θ, the phase-shifting control voltage contains the requiredphase information.

According to this operational principle, the LO signal phase is shifteduntil the IF signal is equal to 0, such that the phase detector outputis independent of the fluorescence pulse amplitude. Because the voltagecontrolled phase shifters have much shorter response times thandividers, as presented above and as shown in FIG. 2, the presentembodiment of the invention (as shown schematically in FIG. 4) canprovide faster phase measurements than are obtainable by a divider-basedphase detector. Additionally, this embodiment provides more stable phaseinformation because no divider is required, whereby the phase shift isindicative of a fluorescence lifetime of a dye corresponding to aphysical or chemical change in a condition of an analyte, cell orparticle.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the above detaileddescription and following Examples of the present invention. It shouldbe understood, however, that the description and specific examples,while indicating preferred embodiments of the present invention, aregiven by way of illustration and not limitation. Many changes andmodifications within the scope of the present .invention may be dewithout departing from the spirit thereof, and the present inventionincludes all such modifications.

EXAMPLE 1 Lifetime Based Flow Cytometry

The following example utilizes an embodiment of the present invention asdescribed above and in FIGS. 5A and 5B and FIG. 6.

FIGS. 7A-7F illustrate the phase fluorescence lifetime measurement withpulses from 2 micrometer diameter fluorophore dyed particles asFluoresbrite™ particles (available from Polysciences, Inc., Warrington,Pa.) analyzed on a computerized flow cytometer, such as the FACStar™(also available Becton Dickinson Immunocytometry Systems). FIG. 7A showsthe signal from the fluorescence detector, such as a PMT which signal isa 20 MHz signal modulated by the signal amplitude. If the signal in FIG.7A is put through a low pass filter to remove the 20 MHz component, theresult is the standard fluorescence amplitude signal, FIG. 7B, whichamplitude is normally detected in a flow cytometer. If the signal inFIG. 7A is fed through a high pass filter the result is shown in FIG.7B, which signal can be further analyzed for phase shift relative to areference signal used to modulate the laser light source, such that thephase shift corresponds to the fluorescent lifetime of the fluorophoreassociated with the cell or particle in the flow chamber.

Before analyzing for the phase shift and or modulation, the signal isfed through a limiter which provides a flat-topped signal during thepeak of the signal, as shown in FIG. 7D. The flat-topped region orvoltage level of FIG. 7D provides a phase measurement that is relativelyindependent of the amplitude of the unlimited raw signal in FIG. 7A.

To measure the phase shift of the detector output signal relative to thereference signal, the limited signal from FIG. 7D is mixed with thereference signal in FIG. 7E. The reference signal in FIG. 7E is alsoused to modulate the laser beam. If there is little or no phase shift,such a short fluorescence lifetime will provide a limited signal (FIGS.7A, 7C and 7D) that is almost exactly in phase with the referencesignal, FIG. 7E, e.g., where the fluorophore used is provided inFluoresbrite™ particles.

The output of the phase-detector, e.g., can be proportional to cos(p),where p is the phase angle. If p=0, there is no phase shift since thefluorescent lifetime zero or is so short as to not be detectable, andthe output of the phase detector is maximal, cos(0)=1. As the lifetimeincreases, the phase angle increases, and the output voltage of thephase detector decreases. Longer lifetimes produce phase detector outputsignals of smaller amplitude. Phase detector output signals for theFluoresbrite™ particles are shown in FIG. 7F. By use of a second mixer,a 90° phase shift, and a ratio circuit, these signals can be used todetermine the phase angle.

To determine the phase detector response as a function of thefluorescence signal amplitude, particles were run on a FACStar™ flowcytometer and the PMT voltage was varied while data was being taken. Foran ideal phase detector, the phase output would be independent of thefluorescence intensity.

FIGS. 8 and 9 show results wherein samples contained a mixture of 2micrometer Fluoresbrite™ particles and calf thymocyte nuclei stainedwith ethidium bromide (EB). DNA stained with EB is reported to have afluorescence lifetime of 24 ns. The x-axis is the standard fluorescencesignal amplitude, while the Y-axis is the output of the phase detector.In FIG. 8, the filter used for the PMT passed only laser light scatteredby the sample particles having no phase lag and thus maximum phasedetector output. This FIG. 8 reflects the known dependence of the PMTtime response on voltage, and reveals the direction of the phase scale.

FIG. 9 shows the result obtained when a 515 nm high pass filter is usedto pass fluorescence output signals from both Fluoresbrite™ particlesand the EB-nuclei to the PMT. The EB-stained nuclei have a lower phasesignal than the Fluoresbrite™ particles. Even for low signal intensitieswhere the phase detector response is reduced, the particles aredistinguished from the nuclei by using correlated phase/amplitude data.In these early measurements, the phase angle scale is arbitrary indirection and magnitude. Also, the change in phase with amplitude,accomplished here by changing the PMT voltage, is known for thetime-response of PMT's. During a phase or modulation measurement the PMTvoltage would be constant.

FIG. 10 shows the phase versus amplitude dot plot of the particle/nucleimixture at a fixed PMT voltage. Particle doublets have nearly the samefluorescence amplitude as the G0/G1 nuclei but are easily resolved bytheir phase (fluorescence lifetime) difference.

As shown by the above-described data, the present invention provides fordetection and characterization of cell component structure andcomposition based on changes in phase angle or modulation of signalscorresponding to changes in lifetimes of at least one fluorophoreassociated with cells or particles used in a flow cytometer and methodsaccording to the present invention.

What is claimed is:
 1. A method of determining, in a flow cytometricenvironment, a fluorescence lifetime of at least one fluorophoreassociated with a cell or particle, comprising the steps of:directing astream of a plurality of said cells or particles past an observationpoint; modulating a light source at a frequency of an input signal toproduce modulated exciting light having a wavelength capable of excitingsaid at least one fluorophore; irradiating at the observation point saidcell or particle with said modulated light to produce emitted light fromsaid at least one fluorophore; detecting said emitted light andproducing a corresponding modulated emission signal; and comparing saidinput signal and said modulated emission signal to produce an outputsignal corresponding to a difference between the phase and/or modulationof said modulated emission signal and said input signal, wherein saidoutput signal is (a) indicative of a value of the fluorescence lifetimeof said at least one fluorophore and (b) is independent of the intensityof the emitted light, and wherein said detecting step further comprisesadjusting said output signal to reduce the effect of autofluorescence.2. A method of determining, in a flow cytometric environment, afluorescence lifetime of at least one fluorophore associated with a cellor particle, comprising the steps of:directing a stream of a pluralityof said cells or particles past an observation point; modulating a lightsource at a frequency of an input signal to produce modulated excitinglight having a wavelength capable of exciting said at least onefluorophore; irradiating at the observation point said cell or particlewith said modulated light to produce emitted light from said at leastone fluorophore; detecting said emitted light and producing acorresponding modulated emission signal; and comparing said input signaland said modulated emission signal to produce an output signalcorresponding to a difference between the phase and/or modulation ofsaid modulated emission signal and said input signal, wherein saidoutput Signal is (a) indicative of a value of the fluorescence lifetimeof said at least one fluorophore and (b) is independent of the intensityof the emitted light, and wherein the comparing step comprisesdividingsaid input signal into first and second signals; dividing said emissionsignal into third and fourth signals; placing said first signal intophase quadrature with respect to said second signal; mixing said firstand third signals and said second and fourth signals, respectively, togenerate fifth and sixth signals, respectively, that are in phasequadrature; and determining the ratio of said fifth and sixth signals inorder to generate the value which is indicative of the fluorescencelifetime of said at least one fluorophore but which value is independentof intensity of said emitted light.
 3. The method of claim 2, furthercomprising, prior to said mixing step, amplifying and limiting each ofsaid first, second, third and fourth signals, respectively, by acorresponding amplifier and limiting circuit.
 4. The method of claim 3,further comprising, prior to said determining step, filtering each ofsaid fifth and sixth signals, respectively, by a corresponding low passfilter.
 5. The method of claim 4, wherein said determining stepcomprises peak detecting said fifth and sixth signal after saidfiltering.
 6. A method of determining, in a flow cytometric environment,a fluorescence lifetime of at least one fluorophore associated with acell or particle, comprising the steps of:directing a stream of aplurality of said cells or particles past an observation point;modulating a light source at a frequency of an input signal to producemodulated exciting light having a wavelength capable of exciting said atleast one fluorophore; irradiating at the observation point said cell orparticle with said modulated light to produce emitted light from said atleast one fluorophore; detecting said emitted light and producing acorresponding modulated emission signal; and comparing said input signaland said modulated emission signal to produce an output signalcorresponding to a difference between the phase and/or modulation ofsaid modulated emission signal and said input signal, wherein saidoutput signal is (a) indicative of a value of the fluorescence lifetimeof said at least one fluorophore and (b) is independent of the intensityof the emitted light, and wherein the comparing step comprisesdividingsaid emission signal into first and second signals; filtering (a) saidfirst signal by a high pass filter, followed by amplifying and splittingsaid first signal into a third signal and a fourth signal and (b) saidsecond signal with a low pass filter; amplifying and limiting said inputsignal and limiting said third signal, respectively, by passing saidinput signal through a signal amplifier set at a preset level and alimiting circuit, and by passing said third signal through a limitingcircuit; then mixing said third signal and said input signal,respectively, to generate a fifth signal; and determining the ratio ofsaid second and fourth signals as a ratio signal; and comparing saidratio signal and said fifth signal in order to generate a value which isindicative of the fluorescence lifetime of said at least one fluorophorebut which value is independent of the intensity of the emitted light. 7.The method of claim 6, wherein said input signal is provided by oneselected from(a) receiving said modulated light by a photomultipliertube and producing said input signal; and (b) a frequency generator setat said frequency.
 8. A flow cytometer operative to measure afluorescence lifetime of at least one fluorophore associated with a cellor a particle, comprising:a flow chamber for directing a plurality ofcells or particles past an observation point; a light source modulatedby a frequency of an input signal for irradiating, at said observationpoint, said cell or particle with modulated light having a wavelengthcapable of exciting said at least one fluorophore to produce emittedlight; a photodetector for detecting said emitted light and producing acorresponding emission signal; and a phase/modulation detector forgenerating an output signal corresponding to a difference between thephase and/or modulation of said emission signal and said input signal,wherein said output signal is (a) indicative of a value of thefluorescence lifetime of said at least one fluorophore and (b) isindependent of the intensity of the emitted light, and wherein saidapparatus further comprises:a first power splitter for dividing saidinput signal into first and second signals having the same amplitude andfrequency; a second power splitter for dividing said emission signalinto third and fourth signals, said signals having an equal amplitudeand identical frequency; a first phase shifter for providing apredetermined phase shift to said first signal; first and second mixers,said first mixer receiving said first signal of said first powersplitter and said third signal from said second power splitter andgenerating a fifth signal, said second mixer receiving said secondsignal and said fourth signal and generating a sixth signal; a ratiounit for receiving said fifth signal and said sixth signal from saidfirst mixer and said second mixer, respectively, and generating theratio of said fifth signal and said sixth signal as said output signal.9. The apparatus of claim 8 further comprising:first, second, third andfourth amplifiers and limiters, respectively, for amplifying andlimiting each of said first, second, third and fourth signals,respectively, prior to being fed into said first and second mixers. 10.The method of claim 9 further comprising:first and second low passfilters, respectively, for filtering each of said fifth and sixthsignals prior to said generating of said ratio.
 11. The method of claim8, further comprising,first and second peak detectors for peak detectingsaid fifth and sixth signals, respectively, after said filtering.
 12. Aflow cytometer operative to measure a fluorescence lifetime of at leastone fluorophore associated with a cell or a particle, comprising:a flowchamber for directing a plurality of cells or particles past anobservation point; a light source modulated by a frequency of an inputsignal for irradiating, at said observation point, said cell or particlewith modulated light having a wavelength capable of exciting said atleast one fluorophore to produce emitted light; a photodetector fordetecting said emitted light and producing a corresponding emissionsignal; and a phase/modulation detector for generating an output signalcorresponding to a difference between the phase and/or modulation ofsaid emission signal and said input signal, wherein said output signalis (a) indicative of a value of the fluorescence lifetime of said atleast one fluorophore and (b) is independent of the intensity of theemitted light, and wherein said apparatus further comprises:a firstpower splitter for dividing said emission signal into first and secondsignals, said signals having an equal amplitude and identical frequency;a high pass filter for filtering said first signal; a first amplifierand a second power splitter, respectively, for amplifying and splittingsaid first signal into a third signal and a fourth signal; a first lowpass filter for filtering said second signal; a second amplifier and afirst limiting circuit, respectively, for amplifying and limiting saidinput signal; a second limiting circuit for limiting said third signal;a mixer for mixing said third signal and said input signal,respectively, to generate a fifth signal; a second low pass filter forfiltering ,said fifth signal; and a ratio unit for determining the ratioof said second and fourth signals as a ratio signal; wherein saidphase/modulation detector is fed said ratio signal and said fifth signalto determine said output signal.
 13. The method of claim 12 furthercomprising:a photomultiplier tube for receiving said modulated light andproducing said input signal.
 14. The method of claim 12 furthercomprising:a frequency generator for producing said input signal at saidfrequency.
 15. A flow cytometer operative to measure a fluorescencelifetime of at least one fluorophore associated with a cell or aparticle, comprising:a flow chamber for directing a plurality of cellsor particles past an observation point; a light source modulated by afrequency of an input signal for irradiating, at said observation point,said cell or particle with modulated light having a wavelength capableof exciting said at least one fluorophore to produce emitted light; aphotodetector for detecting said emitted light and producing acorresponding emission signal; and a phase/modulation detector forgenerating an output signal corresponding to a difference between thephase and/or modulation of said emission signal and said input signal,wherein said output signal is (a) indicative of a value of thefluorescence lifetime of said at least one fluorophore and (b) isindependent of the intensity of the emitted light, and wherein saidapparatus further comprises:an input optics system operative to shapeand direct said modulated light to said observation point; and an outputoptics system operative to receive and direct said emitted light ontosaid photodetector.
 16. A flow cytometer operative to measure afluorescence lifetime of at least one fluorophore associated with a cellor a particle, comprising:a flow chamber for directing a plurality ofcells or particles past an observation point; a light source modulatedby a frequency of an input signal for irradiating, at said observationpoint, said cell or particle with modulated light having a wavelengthcapable of exciting said at least one fluorophore to produce emittedlight; a photodetector for detecting said emitted light and producing acorresponding emission signal; and a phase/modulation detector forgenerating an output signal corresponding to a difference between thephase and/or modulation of said emission signal and said input signal,wherein said output signal is (a) indicative of a value of thefluorescence lifetime of said at least one fluorophore and (b) isindependent of the intensity of the emitted light, and wherein saidapparatus further comprises:a variable phase shifter operative to shiftthe phase of said input signal.
 17. The apparatus of claim 16 whereinsaid variable phase shifter is operative to reduce the effect ofautofluorescence.
 18. The apparatus of claim 17 wherein signalscontributed by said autofluorescence are reduced substantially to zero.